A method of cell-free protein synthesis includes taking components involved in biological protein synthesis out of the body and semi-artificially synthesizing a protein using a nucleic acid such as messenger RNA (mRNA) encoding the objective protein, energy sources such as ATP, GTP etc., and amino acid to be a starting material, but without using a cell. When compared to protein synthesis methods employed heretofore using recombinant microorganisms and cultured-cells, this method is advantageous in that (1) a protein that acts negatively on the body (except protein influential on the translation system) can be synthesized relatively easily, (2) the conditions can be determined easily (the use of microorganism etc. generally requires at least about one month for determination of the conditions), (3) unnatural amino acid can be used, and so on. In view of these advantages, it is expected that a method of cell-free protein synthesis will be widely used for various applications where many kinds of proteins need to be synthesized in a short time, production of a protein by a recombinant organism is difficult and so on.
The biomaterial to be used for a method of cell-free protein synthesis is frequently Escherichia coli, reticulocyte from mammal blood or wheat embryo. To date, various methods for preparing or utilizing cell extracts have been constructed. However, the protein synthesis activity of these cell extracts decreases when preserved at a temperature higher than −70° C. At present, therefore, the extract is typically frozen at −70° C. or below and preserved and transported at this temperature. In addition, protein synthesis requires various enzymes and factors of more than about 50 kinds including ribosome and the like, and stable supply of them requires a means to collectively stabilize a mixture of these components.
To meet the request, the technique for cold storage of cell extracts by freeze-drying has been developed in recent years. It is a general practice to use additives to facilitate stabilization by freeze-drying. As a result, trehalose has been reported to stabilize cell extracts during freeze-drying for cell-free protein synthesis (WO9802532). When additives are determined, stability during long-term storage is an important aspect. For confirmation of the stability during long-term storage, a storage stability test is generally done under harsh conditions (preservation at 25° C. or higher and the like), which is considered to well reflect the actual long-term storage stability. In the investigation by the present inventors, however, addition of trehalose failed to prevent degradation of the activity at 37° C., leaving uncertainty in a successful long-term preservation.
As a matter of fact, the storage stability of frozen cell extract for cell-free protein synthesis, particularly stabilization at around −30° C. to −15° C., which is a common preservation and transport temperature, has been scarcely studied heretofore. As the situation stands, there is a demand for additives more effective on the storage stability of cell extract for cell-free protein synthesis.
It is therefore an object of the present invention to provide a composition for cell-free protein synthesis, which is superior in storage stability in a freeze-dry state, and a composition for cell-free protein synthesis, which is superior in storage stability in a frozen state.